This study was conducted to assess the potential effect of Bifidobacterium bifidum ATTC 29521(B. bifidum ) and Bifidobacterium longum ATTC 15707(B. longum) inoculated in yolk sac of developing embryo of broilers at 18 days of incubation on growth parameters, antioxidant and immunological status and gut microflora. Three hundred broiler breeder eggs (Cobb-500) obtained from maternal flock aged 49 weeks were individually weighed at day 18^th of incubation then divided into six equal treatment groups as follow; C- (non-inoculated, negative control); C+ ( (inoculated with sterile distilled water (vehicle), positive control); B.bifidum H (inoculated with 5x109cfu/ ml); B. bifidum  L (inoculated with 1x107cfu/ml); B. longum H (inoculated with 2x109cfu/ ml); and B. longum L (inoculated with 7x107cfu/ ml). Results showed that, at 35 days of age live body weight (LBW), average daily weight gain (ADWG) and feed conversion ratio (FCR) were significantly improved in bacterial inoculation groups compared with control groups. Improvement in antioxidant status was noted at 35 days of age in bacterial injected groups. Serum GSH content was insignificant increased while MDA content was significantly decreased in B. bifidum  H and B. longum H groups. Moreover, SOD activity was significantly increased in B. bifidum  H and B. longum L groups. Immunological parameters were also enhanced with inoculation of bifidobacteria strains. Serum concentration of total immunoglobulins, IgG and IgM were significantly increased with bifidobacteria inoculation compared with control groups. Furthermore, antibody titer against NDV was increased during the secondary response in injected groups than control groups. All examined microbiological parameters were also significantly affected by in ovo inoculation with bifidobacteria strains. A significant reduction was recorded for total bacteria, total coliform, fecal coliform and Salmonella spp. counts, however, bifidobacteria and total lactic acid bacteria were significantly increased in inoculation groups than controls. It is concluded that injection of B. bifidum  and B. longum enhances growth, antioxidant and Immunological status, activities of gut microflora and reducing the pathogenic bacteria of Cobb-500 broiler chickens. The recommended dose of in ovo inoculation is 5x10⁹cfu / ml of B. bifidum  and 7x10⁷cfu / ml of B. longum.